Abstract 2433 Poster Board II-410 We previously developed robust methods to purify human hematopoietic progenitors based on their high expression of aldehyde dehydrogenase (ALDHbr cells). These cells are enriched with both short- and long-term NOD/SCID repopulating cells. One early clinical trial suggested that augmenting a conventional cord blood transplant (CBT) with ALDHbr cells accelerated both neutrophil and platelet engraftment. We now describe experimental models for these clinical transplants, performed in two immunologically disparate strains of NOD/SCID mice. In the clinical study, pediatric patients first received 80% of an unmanipulated cord blood (CB) graft. 4 hours later they received the ALDHbr cells purified from the remaining 20% of the graft. In the experimental model, the mice were divided among three cohorts. Some mice were transplanted with 4,000 purified ALDHbr cells, alone. Other mice were transplanted with total CB using a cell dose that contained 4,000 ALDHbr cells. In the final group, mice first received the same dose of unmanipulated CB and, after 4 hours, 4,000 purified ALDHbr cells were also administered. After 4 weeks, the mice were sacrificed to determine their levels of human hematopoietic chimerism. When these transplants were performed using NOD/SCID-IL2Rγnull (NSγ) mice, the ALDHbr cells demonstrated strong short-term engraftment to the bone marrow (12 ± 4.9%; n = 5) that was characterized by human CD19+ B cells and CD33+ myeloid cells. The latter included CD15+ cells that indicate neutrophil engraftment. In addition, the peripheral blood of these mice contained low levels of human CD41+ CD61+ platelets. Unmanipulated CB also engrafted the bone marrow of NSγ mice (7.4 ± 4.7%; n = 8); however, >95% of the human cells appeared to be mature CD3+ T cells. Engraftment by either B cells or myeloid cells was consistently low to undetectable. Similarly, human platelets were not detected in the peripheral blood. When NSγ mice were transplanted first with total CB and subsequently with purified ALDHbr cells, the level of engraftment to the bone marrow increased >2-fold over what had been observed in mice transplanted with CB alone (18.9 ± 9.3%; n = 10; P = 0.006). However, nearly all of the human cells present within the bone marrow were T cells, as had been observed in animals that received only unmanipulated CB. In addition, human platelets were not observed in the peripheral blood of NSγ mice that had received both CB and ALDHbr cells. When similar transplants were performed using NOD/SCID-β2-microglobulinnull (NSβ) mice, the ALDHbr cells again demonstrated strong short-term engraftment to the bone marrow (5.6 ± 4.7%; n = 5) that was characterized by human CD19+ B cells and CD33+ myeloid cells. The peripheral blood of these mice also contained low levels of human platelets. In contrast, total CB achieved only very low engraftment to the bone marrow of NSβ mice (0.32 ± 0.19%; n = 4) and human platelets were not detected in the peripheral blood. However, when NSβ mice first received total CB which was augmented 4 hours later with purified ALDHbr progenitors, the level of human hematopoietic chimerism in the bone marrow increased >10-fold (4.3 ± 1.9%; n = 5), with engraftment of human B cells and myeloid cells. The co-transplanted NSβ mice also demonstrated low-level engraftment of human platelets in the peripheral blood. Continuing studies will resolve the relative contributions of the ALDHbr cells and the unmanipulated CB by using two HLA-matched (6/6), but sex-mismatched, CBs. In total, these studies confirmed that ALDHbr progenitors by themselves provided efficient short-term myeloid engraftment in both NOD/SCID strains. This was in contrast to what was observed after transplantation of bulk CB, which by itself did not provide efficient myeloid engraftment in either strain. Finally, in both mouse strains, ALDHbr cells altered the outcome of CB transplants. In NSγ mice, ALDHbr progenitors appeared to facilitate either the engraftment or proliferation of mature CB T cells. Most importantly, the studies in NSβ mice strongly suggested that ALDHbr progenitors directly augment short-term myeloid and platelet engraftment by total CB. The latter studies in particular mirror the experience of early clinical CBT studies that use the same strategy. Disclosures: Storms: Aldagen, Inc: Equity Ownership, Patents & Royalties. Gentry:Aldagen, Inc: Employment. Balber:Aldagen, Inc: Employment, Equity Ownership. Kurtzberg:Aldagen, Inc: Research Funding.

In previous work, we fractionated CD34+ umbilical cord blood (UCB) progenitors to purify cells that express high levels of aldehyde dehydrogenase (ALDH). The ALDHbr CD34+ cells were enriched with progenitors that engraft NOD/SCID mice in both short-term (6–8 week) and long-term (>18 week) transplants. In contrast, ALDHneg CD34+ progenitors were not a significant source of NOD/SCID repopulating cells. These data strongly imply that transplantable human progenitors express ALDH; however, one shortcoming for that work remains that human hematopoietic development in NOD/SCID mice is limited to the myeloid and B-lymphoid lineages. Because the pace of T cell engraftment is a critical clinical concern, we have adopted the use of the NOD/SCID-IL2Rγnull mouse xenograft transplant model. In the current study, 20 mice were transplanted with lineage-depleted ALDHbr CD34+ progenitors at doses that ranged from 3,000 to 30,000 cells. In control studies, similar doses of ALDHneg CD34+ cells did not provide long-term engraftment. So that we might characterize the behavior of individual UCB, each transplant graft was derived from a single UCB. In addition, each UCB was assayed in multiple different mice. Twelve of 13 mice that survived until 21 weeks post-transplant demonstrated multiple-lineage human hematopoietic engraftment. Human T, B, NK and myeloid cells were detected in the bone marrow and spleen. In addition, in 9 mice human T cells were detected in the thymus. Furthermore, 7 mice had sufficient thymus tissue to measure T cell Receptor Excision Circles (TREC). By that assay, 3 of those mice demonstrated active human T cell rearrangements. As anticipated, the level of engraftment to all tissues was cell dose-dependent. One strength for this xenograft model was that engraftment could be monitored over time within the peripheral blood. Human B cells engrafted within 7 weeks post-transplant and were detected in 17 of 19 mice (≥ 5 B cells/μL). At 16 weeks post-transplant, 15 of 18 mice maintained detectable B cells. In contrast, human T cells emerged later, beginning at 13 weeks post-transplant. However, even by 16 weeks post-transplant, T cells were only detectable in the peripheral blood of 6 of 18 mice (≥ 5 T cells/μL). These studies provided evidence that ALDHbr CD34+ cells establish both short- and long-term hematopoiesis in NOD/SCID-IL2Rγnull mice. The emergence of T cells was the most stringent test for long-term engraftment.

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDH(br) CD34+, ALDH(neg) CD34+, and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.

Adipose-derived stromal (AdS) cells are multipotent mesodermal progenitors with developmental potentials similar to those ascribed to bone marrow-derived stromal (MdS) cells. The bone marrow mesenchyme supports hematopoietic stem cells and progenitors in vivo and in vitro. The AdS are readily accessible from subcutaneous fat but have not been fully characterized for hematopoietic support. We now directly compare the relative capacities of allogeneic AdS and MdS to support hematopoiesis. In total, 4 preparations of AdS and 4 preparations of MdS were used to complete these studies. Support for primitive hematopoietic progenitors was monitored in 5-week long-term culture (LTC) assays. CD34+ CD38neg cells were purified from umbilical cord blood (UCB, n = 8) to establish duplicate cultures with 100 hematopoietic progenitors per well in standard Dexter-type media. LTC cultures on MdS gave rise to 67.1 ± 19.8 CFU after 5 weeks. Parallel cultures on AdS gave rise to 26.6 ± 11.4 CFU. These frequencies approach statistical significance (p = 0.054). Importantly, however, 30% of the cultures initiated on AdS failed to support LTC. Thus, 100 CD34+ CD38neg cells may define a limit-dilution for LTC activity on AdS cells. In contrast, all cultures initiated on MdS supported LTC. In shorter-term assays, parallel cultures were established using 10,000 CD34+ linneg UCB cells for 12 days. The cultures were established in DMEM/F12 media containing 10% serum. No exogenous cytokines were used. The results are tabulated below. AdS and MdS supported similar expansions of total hematopoietic (CD45+) cells. However, cultures established on MdS maintained higher percentages of CD34+ cells over the 12 day time course. Not surprisingly, there was a strong association between the percentages of CD34+ cells and the frequencies of CFU present at day 12 (AdS, r = 0.81; MdS, r = 0.85). However, without exogenous cytokines these stroma maintained but did not expand the total content of CFU. Finally, both AdS and MdS encouraged the outgrowth of lymphoid progenitors as monitored by the presence of CD7+ CD34neg cells as well as by the growth of NK (CD56+) cells in secondary cultures. Higher percentages of CD7+ cells were noted on AdS than on MdS. In total, these data suggest that AdS and MdS provide similar hematopoietic support, although subtle distinctions were noted. These studies provide a model that can be manipulated to optimize hematopoietic expansion ex vivo. TableHematopoietic Support in Short-term CultureCD45+ Cell ExpansionPercent CD34+, Day 12CFU/100 Input CellsCFU ExpansionPercent CD7+, Day 12AdS3.78 ± 1.1 fold17.4 ± 5.1 %12.2 ± 2.8 CFU0.90 ± 0.3 fold28.6 ± 5.6 %MdS3.4 ± 1.3 fold28.7 ± 6.5 %16.3 ± 3.7 CFU1.07 ± 0.27 fold19.5 ± 4.2 %P0.1270.0020.0620.3660.003n88886Values reported are the average ± SEM, P values are by t-test. Open table in a new tab Adipose-derived stromal (AdS) cells are multipotent mesodermal progenitors with developmental potentials similar to those ascribed to bone marrow-derived stromal (MdS) cells. The bone marrow mesenchyme supports hematopoietic stem cells and progenitors in vivo and in vitro. The AdS are readily accessible from subcutaneous fat but have not been fully characterized for hematopoietic support. We now directly compare the relative capacities of allogeneic AdS and MdS to support hematopoiesis. In total, 4 preparations of AdS and 4 preparations of MdS were used to complete these studies. Support for primitive hematopoietic progenitors was monitored in 5-week long-term culture (LTC) assays. CD34+ CD38neg cells were purified from umbilical cord blood (UCB, n = 8) to establish duplicate cultures with 100 hematopoietic progenitors per well in standard Dexter-type media. LTC cultures on MdS gave rise to 67.1 ± 19.8 CFU after 5 weeks. Parallel cultures on AdS gave rise to 26.6 ± 11.4 CFU. These frequencies approach statistical significance (p = 0.054). Importantly, however, 30% of the cultures initiated on AdS failed to support LTC. Thus, 100 CD34+ CD38neg cells may define a limit-dilution for LTC activity on AdS cells. In contrast, all cultures initiated on MdS supported LTC. In shorter-term assays, parallel cultures were established using 10,000 CD34+ linneg UCB cells for 12 days. The cultures were established in DMEM/F12 media containing 10% serum. No exogenous cytokines were used. The results are tabulated below. AdS and MdS supported similar expansions of total hematopoietic (CD45+) cells. However, cultures established on MdS maintained higher percentages of CD34+ cells over the 12 day time course. Not surprisingly, there was a strong association between the percentages of CD34+ cells and the frequencies of CFU present at day 12 (AdS, r = 0.81; MdS, r = 0.85). However, without exogenous cytokines these stroma maintained but did not expand the total content of CFU. Finally, both AdS and MdS encouraged the outgrowth of lymphoid progenitors as monitored by the presence of CD7+ CD34neg cells as well as by the growth of NK (CD56+) cells in secondary cultures. Higher percentages of CD7+ cells were noted on AdS than on MdS. In total, these data suggest that AdS and MdS provide similar hematopoietic support, although subtle distinctions were noted. These studies provide a model that can be manipulated to optimize hematopoietic expansion ex vivo. TableHematopoietic Support in Short-term CultureCD45+ Cell ExpansionPercent CD34+, Day 12CFU/100 Input CellsCFU ExpansionPercent CD7+, Day 12AdS3.78 ± 1.1 fold17.4 ± 5.1 %12.2 ± 2.8 CFU0.90 ± 0.3 fold28.6 ± 5.6 %MdS3.4 ± 1.3 fold28.7 ± 6.5 %16.3 ± 3.7 CFU1.07 ± 0.27 fold19.5 ± 4.2 %P0.1270.0020.0620.3660.003n88886Values reported are the average ± SEM, P values are by t-test. Open table in a new tab Values reported are the average ± SEM, P values are by t-test.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

Hematopoietic stem cells have been identified as multipotent cells that give rise to all adult hematopoietic lineages. Although the hematopoietic lineage is derived from the mesodermal germ layer in the embryo, recent data suggest that bone marrow cells with an antigenic profile consistent with that of hematopoietic stem cells can also differentiate to cell types of the endodermal lineages, such as hepatocytes. However, the molecular mechanisms associated with these events are entirely unknown. For decades, alpha-fetoprotein (AFP) has been used as a differentiation marker for endodermal cells, because it was thought that the transcription of AFP mRNA is tightly regulated in a developmental and tissue-specific process. In this report we describe two new variant forms of AFP transcripts in human hematopoietic progenitors that are not expressed in mature cells. The variant AFP (vAFP) cDNA sequences isolated from a multipotent hematopoietic cell line, K562, revealed that the vAFP differed from the authentic transcript, consisting of 15 exons, by replacing exon 1 of AFP with one or two exons located in the 5'-untranslated region of the AFP gene. In addition to the K562 cell line, vAFP transcripts were detected in normal bone marrow, thymus, and brain but were not detected in normal spleen, intestine, liver, or the hepatocellular carcinoma cell line, HepG2. This suggests expression in normal hematopoietic progenitors. This hypothesis was confirmed by the finding that CD34(+)Lin(-) hematopoietic progenitor cells purified from cord blood by flow cytometric sorting also expressed the variant transcripts. These results suggest that some hematopoietic progenitors are in a state that permits them to express certain types of transcripts that have been considered unique to endoderm.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue‐derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue‐derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49d, CD 49e, CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow‐derived stromal cells, we did not detect the STRO‐1 antigen on human adipose tissue‐derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPα and PPARδ, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue‐derived and human bone marrow‐derived stromal cells could have broad implications for human tissue engineering. © 2001 Wiley‐Liss, Inc.

A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34(-) cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34(+) cells but also contained a large population of CD34(-) cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin(-) UCB) contained CD34(+) and CD34(-) cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34(+)Lin(-) SP cells were CD38(dim)HLA-DR(dim)Thy-1(dim)CD45RA(-)CD71(-) and were enriched for myelo-erythroid precursors. In contrast, the CD34(-)Lin(-) SP cells were CD38(-)HLA-DR(-)Thy-1(-)CD71(-) and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7(+)CD11b(+)CD45RA(+), as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7(+)CD34(-)Lin(-) UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin(-) UCB SP contains both CD34(+) multipotential stem cells and a novel CD7(+)CD34(-)Lin(-) lymphoid progenitor. This observation adds to the growing body of evidence that CD34(-) progenitors exist in humans.

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity, we developed a fluorescent substrate for ALDH, termed BODIPY aminoacetaldehyde (BAAA), and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells, 40-90% pure for CD34(+)CD38(lo/-) cells, and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together, these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well.